Sickle cell anemia (SCA) is one of the most common inherited hematological diseases, and caused by a single amino acid substitution in the 2-globin chain of hemoglobin (Hb). The long-range goal of our research program is to develop a safe and effective gene transfer approach for treating SCA patients. To accomplish our objective, we propose an R21/R33 phased innovation grant application using a novel non- viral gene therapy that will be optimized in transgenic sickle mice and ultimately tested in patients with SCA. The R21 phase is designed to establish proof-of-concept that can be implemented for the R33 developmental phase. The proposal will test the hypothesis that a 2-globin minigene can transform CD34+ stem cells from human SCA patients and generate persistent erythroid-specific expression of the transgene for use in human mini-transplants and will improve the clinical phenotype in patients. In the R21 exploratory phase, Specific Aim 1 is designed to optimize the Sleeping Beauty transposon (SB-Tn) and transfection efficiency in mouse bone marrow (BM) and hematopoietic stem cells (HSCs). In Specific Aim 2, we will examine transformed sickle transgenic mouse BM and phenotypic c-kit+Thy1+Lineage(Lin)-Sca1+ (KTLS) cells for long-term culture initiating cells (LTC-ICs), erythroid-specificity and persistence of IHK- driven transgene expression in LTC-ICs, and SB-Tn transposition sites in ex vivo culture. With successful completion of the R21 studies, we will implement the R33 development phase and examine the biological response of transgenic sickle mice to stem cell gene therapy by examining red cell sickling, Hb expression, hemolysis, oxidative stress, vascular inflammation and vaso-occlusion. Specific Aim 1 of the R33 phase is designed to test the erythroid-specific 2-globin transgene in sickle human progenitor cells derived from the peripheral blood CD34+ population. During this stage, we will begin the process of transferring the vector generation and cell therapy processes from research status to GMP status, taking advantage of the Cancer Centers Translational Therapy Core system. In Specific Aim 2, we will carry out non-ablative mini-transplant of autologous, engineered HSCs in NOD-SCID and NY1DD sickle mice. In Specific Aim 3, after obtaining all necessary regulatory and oversight approvals, we will test the final construct in five adult human sickle patients, using autologous engineered CD34+ cells from mobilized (but not by G-CSF) peripheral blood in a non-ablative mini-transplant protocol. Endpoints tested will include all those necessary to monitor and ensure subject safety, as well as those related to long-term assessment of engineered engraftment stability and efficacy. All vector and cell products at this stage will have been produced and quality controlled through our institution's NIH approved GMP Minnesota Molecular and Cellular Therapy facility. Our ultimate goal is to show that the 2-globin transgene can transform CD34+ stem cells from SCA patients and sustain an improved sickle phenotype after reduced intensity autologous transplantation of the gene-modified HSCs. PUBLIC HEALTH RELEVANCE: Sickle cell anemia (SCA) is one of the most common inherited hematological disorders and affects over 72,000 Americans and thousands of others worldwide (NIH Publication No. 96-4057). This relentless disease, manifested by hemolytic anemia and painful vaso-occlusive crises leading to organ damage, has a significant impact on the quality and length of life, costing millions in care and lost economic productivity. Therapies including hydroxyurea, exchange transfusion, prophylactic antibiotics, and stem cell transplants have improved the outlook for these patients. However, despite these advances, many patients continue to experience painful crises, acute chest syndromes, and strokes, all of which take their accumulative toll on the quality and length of life. To address these issues, we propose a non-viral transfer of the normal human 2-globin gene into hematopoietic stem cells and re-introduction of these genetically altered stem cells into bone marrow. These studies will initially be carried out in transgenic sickle cell mice;and if successful the techniques will be applied to stem cells from human sickle cell patients. We will use a unique non-viral gene therapy approach to avoid the potential side effects associated with viral vector gene transfer. The ultimate goal of this research project is to develop and evaluate in patients an innovative gene therapy approach that has the potential to treat and correct SCA.